CN117402911A

CN117402911A - Application of interfering transcription factor PtoMYB240 of populus tomentosa in improving biomass of populus tomentosa

Info

Publication number: CN117402911A
Application number: CN202311536625.5A
Authority: CN
Inventors: 罗克明; 宋琴; 赵政杰; 孔令飞; 张萌
Original assignee: Western Chongqing Science City Germplasm Creation Science Center
Current assignee: Western Chongqing Science City Germplasm Creation Science Center
Priority date: 2023-11-11
Filing date: 2023-11-17
Publication date: 2024-01-16

Abstract

The invention discloses an application of interfering expression of a transcription factor PtoMYB240 of populus tomentosa in improving biomass of populus tomentosa, and PtoMYB240RNAi transgenic plants are obtained by introducing an RNAi plant vector containing PtoMYB240 into populus tomentosa. The results showed that the expression level of PtoMYB240 was reduced by 0.5-fold and 10-fold, respectively, compared to the wild type. Under the condition of normal soil moisture, the transgenic poplar has little difference in plant height compared with WT, but is thicker and larger in terms of stem thickness and leaf size; therefore, after the PtoMYB240 expression of the populus tomentosa is interfered, the whole biomass of the populus tomentosa can be effectively enhanced, and more wood raw materials are provided for the aspects of building, papermaking and biological energy.

Description

Application of interfering transcription factor PtoMYB240 of populus tomentosa in improving biomass of populus tomentosa

Technical Field

The invention relates to the field of biotechnology, in particular to an application of interfering expression of a transcription factor PtoMYB240 of populus tomentosa in improving biomass of populus tomentosa.

Background

Biomass or carbon reserves, total amount of organic matter stored per unit area. This index is typically used to quantify the productivity and carbon sequestration capacity of forest ecosystems. Biomass partitioning reflects the adaptation of plants (or groups of plants) to different environments, typically expressed as the ratio of the underground biomass of the plant to the above-ground biomass (i.e., root cap ratio). At present, forest biomass and distribution thereof have been widely studied on different forest types, climate areas and geographic scales, and are important indexes for forest resource monitoring. At present, development of breeding work of new varieties of high-biomass woods is urgently needed. However, the traditional tree breeding has a long period and a plurality of difficulties such as difficult breeding. Therefore, the creation of the biomass tree new germplasm by using the genetic engineering technology has wide development prospect.

Poplar (Populus spp.) is one of the most widely planted fast-growing trees worldwide, the trunk is straight, and the trees can be cut and utilized in a shorter period of time, so that the tree is one of the main planted tree species of the global fast-growing forest. In addition, the root system of the poplar is developed, the poplar can prevent wind and fix sand, reduce water and soil loss, and is an ideal tree species for greening forests and cities and villages (Shi Gongming.2009). In addition to having important ecological value, poplar has a wide range of industrial and architectural uses, for example, poplar can be used as a raw material for pulp and paper, as a raw material for fiber boards and plywood, as a material for construction and furniture, and as an important raw material for bioenergy industry (Wu Dingxin et al, 1997; fu Feng et al, 1999; north yellow. 2013).

The populus tomentosa is a special rural tree species in China, and also becomes a widely planted wood tree species in northern areas due to the characteristics of rapid growth, excellent materials and strong adaptability. In recent years, the establishment of genetic transformation systems and gene knockout techniques of populus tomentosa has made it possible to deeply study the regulatory mechanisms of important traits such as wood formation and environmental adaptation (Fan et al 2015; xu et al).

2017). Along with the deep research and analysis of the poplar biomass regulation mechanism, excellent tree species are created by the genetic engineering technology, so that the adaptation capability of the forest to different forest areas is improved, and the method has important scientific significance and economic value.

At present, research on poplar biomass is mainly focused on nitrogenous fertilizers (Wang Guobing.2016, an Yuchao.2023), planting density (peak.2022), soil microorganisms (house phoenix.2020) and the like, and little research on improvement on poplar wood biomass by gene editing is focused on regulation of secondary xylem by MYB240 in MYB transcription families.

MYB family genes are reported to be widely involved in regulating the secondary developmental processes of plants, and several R2R3-MYBs are involved in the regulation of flavonoid biosynthesis. For example, in arabidopsis, MYB115 and MYB134 transcription regulate the accumulation of plant procyanidins and salicylates (Amy Midori James et al., 2017), and the poplar gene PtrMYB152 regulates the synthesis of arabidopsis secondary walls (glucai Wang et al., 2012). MYB96 regulates arabidopsis seed dormancy (Stracke, R, 2007), atMYBR1/AtMYB44 plays a multiple role in ABA signaling, stress response, and leaf senescence (Masrur R Jaradat et al., 2013). These studies indicate that MYB-like transcription factors play an important role in mediating various plant development, but MYB-like transcription factors that regulate overall plant biological yield remain to be further explored.

Disclosure of Invention

Accordingly, one of the objects of the present invention is to provide an application of interfering with the expression of the transcription factor PtoMYB240 of populus tomentosa in increasing the biomass of populus tomentosa; the second object of the present invention is to provide a method for increasing the biomass of aspen.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. the application of interfering the expression of a transcription factor PtoMYB240 of populus tomentosa in improving the biomass of populus tomentosa is provided, wherein the nucleotide sequence of the transcription factor PtoMYB240 is shown as SEQ ID NO. 1.

Preferably, the biomass is stem crude or dry weight.

In the preferred embodiment of the invention, the method for interfering with the expression of the transcription factor PtoMYB240 of populus tomentosa is by using RNAi technology.

Preferably, the method for interfering the expression of the transcription factor PtoMYB240 of the populus tomentosa converts a recombinant vector containing an RNAi fragment of the transcription factor PtoMYB240 into populus tomentosa, and screens transgenic plants.

Preferably, the nucleotide sequence of the RNAi fragment is shown as SEQ ID NO. 2.

2. A method for improving the biomass of white poplar comprises transforming white poplar with recombinant vector containing RNAi fragment of transcription factor PtoMYB240, screening transgenic plant, and obtaining transgenic plant of white poplar with improved biomass.

Preferably, the nucleotide sequence of the RNAi fragment of the transcription factor PtoMYB240 is shown as SEQ ID NO. 2.

Preferably, the transformation is agrobacterium-mediated.

Preferably, the agrobacterium is agrobacterium GV3101.

The invention has the beneficial effects that: the invention provides application of interfering expression of a transcription factor PtoMYB240 of populus tomentosa in improving biomass of populus tomentosa, and PtoMYB240RNAi transgenic plants are obtained by introducing an RNAi plant vector containing PtoMYB240 into populus tomentosa. The results showed that the expression level of PtoMYB240 was reduced by 0.5-fold and 10-fold, respectively, compared to the wild type. Under the condition of normal soil moisture, the transgenic poplar has little difference in plant height compared with WT, but is thicker and larger in terms of stem thickness and leaf size; therefore, after the PtoMYB240 expression of the populus tomentosa is interfered, the whole biomass of the populus tomentosa can be effectively enhanced, and more wood raw materials are provided for the aspects of building, papermaking and biological energy.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1. Creation of PtoMYB240RNAi plants from Populus tomentosa. The PtoMYB240RNAi gene fragment is inserted between BamHI cleavage sites on the pCXSN vector to construct the PtoMYB240RNAi-pCXSN vector.

FIG. 2 identification of PtoMYB240RNAi positive plants from populus tomentosa. A: PCR molecular detection of wild type PtoMYB240RNAi transgenic plants and PtoMYB240RNAi transgenic plants, wherein DNA of the wild type plants and PtoMYB240RNAi transgenic plants is used as a template, and PCR detection is carried out by using vector primers HYG-F and HYG-R; wherein M represents Marker DL2000; WT is a wild plant, L1-L11 is PtoMYB240RNAi plant to be identified; b: analysis of expression level of PtoMYB240RNAi plants of populus tomentosa.

FIG. 3 analysis of phenotype and biomass scale of PtoMYB240RNAi plants from Populus tomentosa. Wherein A: phenotype analysis of wild and transgenic aspen plants under different soil moisture conditions, scale = 10cm; b: the stem thickness of wild and transgenic aspen plants is counted under normal conditions; c: and (5) carrying out dry weight statistics on wild and transgenic aspen plants. The single factor analysis of variance detects significant differences, with different letters representing significant differences.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.

EXAMPLE 1 extraction of Populus tomentosa Total RNA and reverse transcription to cDNA

Soaking the required medicine spoon, mortar, pestle, etc. for RNA extraction with DEPC water overnight, sterilizing at high temperature under high pressure, and oven drying. The RNA extraction reagent was operated using the Axygen kit instructions and the resulting RNA was stored in a-80℃refrigerator for use. The method comprises the following specific steps:

(1) Wrapping the fresh plant tissue with tinfoil paper, and quick freezing in liquid nitrogen;

(2) In a mortar with RNase removed, the sample is fully ground into powder with liquid nitrogen;

(3) Transferring the powder into AG buffer, shaking thoroughly, mixing to obtain slurry, standing at room temperature for 5-10min

(4) Freezing and centrifuging at 4 ℃ at 12000rpm/min for 10min;

(5) Taking the supernatant to a new 1.5mL EP tube, accurately estimating the volume of the supernatant, adding 0.5 times of absolute ethyl alcohol, and uniformly mixing;

(6) Transferring the mixed solution to a spin clum extraction column, and centrifuging at 12000rpm/min for 1min;

(7) Discarding the waste liquid in the collecting pipe, adding 500 mu L of PG buffer into the column, centrifuging at 12000rpm/min for 1min;

(8) Discarding the waste liquid, adding 600 mu L of Wash buffer into the column, and centrifuging at 12000rpm for 30s;

(9) Repeating the step 8 once;

(10) Discarding the waste liquid, centrifuging the empty tube for 12000rpm/min and 1min, and removing the liquid on the filter membrane;

(11) Adding 30-50 mu L RElution buffer in the center of the membrane, standing at room temperature for 2min, centrifuging at 12000rpm/min for 1min to obtain total RNA;

(12) 1 μl of RNA samples were run on agarose gel electrophoresis and the quality of extraction was checked by observing RNA band integrity for 28s and 18 s.

cDNA Synthesis was performed according to PrimeScript ^TM RT reagentKit with gDNA Eraser reverse transcription kit (Takara) instructions, the procedure is as follows:

genomic DNA removal:

(1) 1-7. Mu.L of RNA sample (total amount not exceeding 1. Mu.g) was added depending on the RNA concentration;

(2) Add 2. Mu.L of 5 XDNA digestive enzyme buffer, 1. Mu.L of DNA digestive enzyme;

(3) With the enzyme RNase ddH ₂ O complements the population to 10. Mu.L;

(4) The reaction conditions were 42℃for 2min 30s.

First strand cDNA Synthesis:

to the first reaction system, 4. Mu.L of reverse transcription buffer, 1. Mu.L of oligo dT random primer, 1. Mu.L of reverse transcriptase, 4. Mu.L of RNase-free water was added, and the final reaction system was 20. Mu.L.

The reaction conditions were 37℃for 15min (reverse transcription) -85℃for 5s (termination reaction) -4℃for 5min (temperature reduction preservation).

EXAMPLE 2 design of target Gene-specific primers and PCR amplification to obtain target fragments

The cloned gene sequences in the study are respectively referred to the PtoMYB240 nucleotide sequence (SEQ ID NO. 1) of the populus tomentosa, the PtoMYB240RNAi interference nucleotide sequence of the transcription factor of the populus tomentosa is shown as SEQ ID NO.2, and the pGUSP expression vector sequence (SEQ ID NO. 3). Corresponding primers were designed based on the reference sequence.

(RNAi)PtoMYB240-F：5’-CGGGATCCCAAGAACGTGGTGATCTAATCT-3’(SEQ ID NO.4)；

(RNAi)PtoMYB240-R：5’-CGGGATCCTTTCATCCCAGAGCCATT-3’(SEQ ID NO.5)；

Pto240StufferGUS-F：5’-TGGCTCTGGGATGAAAATCTACCCGCTTC-3’(SEQ ID NO.6)；

Pto240StufferGUS-R：5’-TGGCTCTGGGATGAAATAATCGCCTGTAAG-3’(SEQ ID NO.7)；

Cloning of PtoMYB240 Gene fragment (RNAi) PCR amplification was performed using a PrimeSTAR high-fidelity enzyme (Takara) with cDNA as a template and Pto240StufferGUS gene as a template and pGUSP vector, and the reaction system was as shown in Table 1:

TABLE 1 reaction System

The reaction conditions were set as follows: pre-denaturation at 98 ℃,1min for 30s; denaturation at 98℃for 15 s; annealing at 56 ℃ for 20 s; extending at 72 ℃ for 10 s; a total of 35 cycles; 72 ℃, and 5min of complete extension; cooling and preserving at 16 ℃ for 5 min. The product was detected by 1% agarose gel electrophoresis.

Further, the (RNAi) PtoMYB240 short fragment and the 240StufferGUS fragment obtained by the above amplification were used as templates, and the (RNAi) PtoMYB240-F was used as an amplification primer, and PCR amplification was performed using PrimeSTAR high-fidelity enzyme (Takara), the reaction system being shown in Table 2:

TABLE 2 reaction System

Reagent	Amount
		2 x PrimeSTAR high fidelity enzyme	25μL
(RNAi)PtoMYB240-F	2.5μL
		(RNAi) PtoMYB240 fragment	1 mu L (not more than 100 ng)
240StufferGUS fragment	0.5 mu L (not more than 50 ng)
		ddH ₂ O	21μL
Total	50μL

Since the PCR reaction and the cleavage reaction both contain various components, in order to avoid the influence of each component on the ligation reaction and to reduce the ligation efficiency, the PCR product should be gel-recovered and purified to obtain a pure (RNAi) PtoMYB240-GUS neck-ring-shaped DNA fragment. The target fragment was recovered using the BioFlux gel recovery kit.

Example 3 construction of PtoMYB240RNAi-pCXSN vector and transformation of Agrobacterium GV3101

The overexpression vector (pCXSN) used in this study was a set of TA cloning vector, which was digested with BamHI to generate cohesive ends, and recovered with a gel to allow direct ligation with BamHI-digested DNA fragments.

The cleavage reaction system is shown in Table 3.

TABLE 3 cleavage reaction System

The reaction conditions were set as follows: water bath or metal bath at 37 ℃ for 4-8h. The plasmid and the gel recovery fragment after enzyme digestion are recovered by gel as a carrier skeleton and a connecting fragment for standby, and are stored at-20 ℃.

(2) Recovery and purification of fragments of interest and vector backbones

Because the PCR reaction and the enzyme digestion reaction both contain multiple components, in order to avoid the influence of each component on the ligation reaction and reduce the ligation efficiency, the PCR products and the enzyme digestion products are subjected to gel recovery and purification to obtain pure DNA fragments. The gel recovery of the carrier backbone was performed using a BioFlux gel recovery kit.

(3) Ligation of fragments with vectors

Since the target fragment was amplified using PrimeSTAR high-fidelity enzyme, the end of the product was blunt, and in order to perform TA cloning with pMD19 vector, the amplified fragment was subjected to "A" addition using Taq enzyme, and the reaction system is shown in Table 4:

TABLE 4 reaction System

Reagent	Amount
		High fidelity enzyme amplified blunt end gel recovery product	10μL
2×rTaq Mix	10μL
		Total	20μL

Mixing, placing in a PCR instrument, extending at 72deg.C for 50min, and storing the product at 4deg.C for use.

The DNA fragment added with the "A" is connected with the pMD19 vector, and the Solution I kit of Takara company is connected and used, and the reaction system is shown in the table 5:

TABLE 5 reaction System

Reagent	Amount
		Add "A" fragment	4μL
pMD19 vector	1μL
		Solution I	5μL
Total	10μL

Ligation was performed at 16℃for 4-8h, and the ligation product was used to transform E.coli DH 5. Alpha. Competent cells.

(4) Colony PCR screening of Positive clones

Clone numbers on the plate were counted, and the sterilized gun heads were used to pick up trace cells in the PCR tubes in sequence as amplification templates, and the reaction system was as shown in Table 6. In addition, gene gel is used for recovering fragments and ddH ₂ O is used as a template, and positive and negative controls are respectively set.

TABLE 6 PCR System

The reaction conditions were set as follows: pre-denaturation at 98 ℃,1min for 30s; denaturation at 98℃for 15 s; annealing at 56 ℃ for 20 s; extending at 72 ℃ for 10 s; a total of 35 cycles; 72 ℃, and 5min of complete extension; cooling and preserving at 16 ℃ for 5 min. The product is detected by agarose gel electrophoresis with the concentration of 1%, and can amplify the colony with the same band size as the positive control to be the positive clone.

(5) Positive clone plasmid extraction and enzyme digestion verification

Clones positive for PCR detection were picked up in LB liquid medium containing kanamycin or ampicillin and cultured overnight at 37℃with shaking at 200 rpm/min. Plasmid extraction was performed using the alkaline lysis plasmid miniprep kit from BioFlux company and the plasmids were sent to the company for sequencing. After the sequence is determined and compared, the carrier construction is completed, and the PtoMYB240RNAi sequence is obtained as shown in SEQ ID NO. 2. And (3) performing enzyme digestion on the pMD19-PtoMYB240RNAi recombinant plasmid after successful construction by utilizing BamHI to obtain a DNA fragment of PtoMYB240RNAi with sticky ends, connecting the DNA fragment to the pCXSN linearization fragment after BamHI enzyme digestion to obtain a recombinant plasmid PtoMYB240RNAi-pCXSN (figure 1), and performing agrobacterium transformation operation on the correct recombinant plasmid.

The procedure for transformation of Agrobacterium was as follows:

1) 3 mu L of PtoMYB240RNAi-pCXSN recombinant plasmid is added into 100 mu L of Agrobacterium GV3101 competent cells, and the mixture is gently mixed;

2) Standing on ice for 5-10min, adding into liquid nitrogen, freezing for 1min, immediately adding into 37deg.C water bath for 5min;

3) Adding 800 mu L of YEP liquid culture medium into competent cells in an ultra clean bench, uniformly mixing, and carrying out resuscitating culture for 4 hours at 200rpm/min on a shaking table at 28 ℃;

4) Centrifuging at 3000rpm/min for 10min after resuscitating, discarding 900 μl of supernatant in a super clean bench, mixing the rest 100 μl of bacterial liquid, uniformly coating 100 μl of bacterial liquid on YEP solid culture medium containing 40mg/L rifampicin and 50mg/L kanamycin with sterilized and cooled coating rod, and culturing upside down at 28deg.C for 48 hr;

5) The engineering bacteria named (GV 3101) PtoMYB240RNAi-pCXSN are added with 75% glycerol, quick frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for subsequent genetic transformation experiments.

Example 4 genetic transformation of Populus tomentosa

(1) Two-time activation culture of agrobacterium

1) Streaking (GV 3101) PtoMYB240RNAi-pCXSN strain on a YEP solid medium containing 40mg/L rifampicin and 50mg/L kanamycin, and culturing in a constant temperature incubator at 28℃for 36 hours; single colonies were picked and inoculated into 10ml of yep+rif+kan double-antibody liquid medium;

2) Shaking culture is carried out for 36-48 hours at 28 ℃ and 200rpm/min, so that the concentration of bacterial liquid reaches OD600 = 0.8-1.0;

3) According to 1:1000, sucking 50 mu L of a primary living liquid into 50mL of fresh YEP+Rif+kan double-antibody liquid culture medium, and performing secondary living liquid culture;

4) And (3) carrying out shaking culture at 28 ℃ for 12-16 hours at 200rpm/min to ensure that the concentration of bacterial liquid reaches OD600 = 0.3-0.4 for later use.

(2) Preparation of agrobacterium infection liquid

1) Collecting secondary living liquid by using a 50mL centrifuge tube at 4000rpm/min for 8min, and collecting thalli;

2) Discarding the culture supernatant, re-suspending the agrobacterium with 25mL WPM re-suspension containing AS, and pouring the re-suspension into a sterile glass bottle;

3) The heavy suspension is placed at 28 ℃ and oscillated at 200rpm/min for 1-2 hours, so that the infection activity of the agrobacterium is enhanced.

(3) Leaf disk preparation

1) In an ultra clean bench, burning sterilized scissors, tweezers and a surgical knife handle with an alcohol lamp outer flame for 15 seconds, and cooling for later use;

2) Cutting off 5-6 healthy wild tissue culture seedling leaves by scissors, and placing the leaves into a culture dish. Adding 1/3 volume of sterile water into the dish to keep the leaves moist;

3) And (5) loading the sterile surgical blade into a knife handle, and standing and cooling after flame burning. The blade was cut uniformly into 0.5cm pieces with a blade ² Is provided.

(4) Infestation of the human body

1) Clamping the leaf disc into agrobacterium tumefaciens heavy suspension by using tweezers, and slightly shaking a glass bottle to enable the heavy suspension to uniformly wrap the leaf disc, and carrying out infection for 10min;

2) After the infection is finished, carefully clamping the leaf disc out by using tweezers, putting the leaf disc onto sterile paper, and sucking the redundant invasion solution on the leaf disc;

3) Flatly attaching leaf discs to a co-cultivation flat plate, placing the leaf discs in a cassette, and performing dark cultivation at 25 ℃ for 36-48h.

(5) Selective cultivation of leaf discs

1) After the dark culture is finished, selecting proper plant resistance according to the carrier, and preparing a selection medium containing antibiotics;

2) Leaf discs were transferred to selection medium in an ultra clean bench to induce callus. Every seven days, the leaf disk is replaced to a new culture medium, and the replacement is continued for 3-4 weeks until white or light yellow callus grows out from the edge of the leaf disk. The whole process is cultivated in a cassette at 25 ℃.

(6) Callus induced germination

Leaf discs from which callus grows were transferred to germination medium containing the corresponding antibiotic, and the medium was changed once a week by light culture at 25℃for 5-6 weeks at 8000 Lux. During the period, the callus can fully grow and expand, and about week 5, the bud point can grow on the callus, and cluster buds can grow.

(7) Cluster bud induced rooting

When the cluster buds grow to about 3-5cm, cutting off the buds with sharp scissors, inserting the cluster buds into a rooting medium with forceps, and carrying out illumination culture at the temperature of 25 ℃ for 7-10 days at the temperature of 8000Lux to obtain rooting seedlings. The plant is a candidate transgenic plant, and the soil culture can be transplanted after the subsequent identification is positive.

Example 5 PCR molecular characterization of PtoMYB240RNAi-pCXSN transgenic plants

(1) DNA extraction of wild-type and PtoMYB240RNAi-pCXSN transgenic aspen

And respectively selecting 10-15 transgenic resistant regenerated plants, and extracting the genome DNA of the populus tomentosa. The method comprises the following steps:

1) Preparing CTAB buffer solution, and preheating in a water bath kettle at 65 ℃ for later use;

2) About 0.5g of leaves of wild type and PtoMYB240RNAi-pCXSN transgenic aspen are taken, ground into powder in liquid nitrogen, added into 500 mu L of the preheated CTAB extract and uniformly mixed;

3) The mixture is stirred for three times at intervals (gently) in a water bath at 65 ℃ for 45 min.

4) After the water bath is finished, cooling to room temperature, adding equal volume of chloroform-isoamyl alcohol (24:1), gently reversing, mixing, and flatly placing and emulsifying for 10min. Centrifuging at 4 ℃ at 12000rpm/min for 10min;

5) Sucking the supernatant into a new sterile centrifuge tube, adding isopropyl alcohol precooled at the temperature of-20 ℃ in equal volume, and reversing and uniformly mixing to obtain white flocculent precipitate;

6) Centrifuge at 12000rpm/min at 4℃for 10min. The supernatant was removed, the precipitate was rinsed twice with 500ml of 75% (V/V) ethanol, and again with 500ml of absolute ethanol, and the liquid was removed. Drying the precipitate in a rotary evaporator at 37 ℃ until translucency appears;

7) Dissolving the precipitate with 25 mu L of sterile water to obtain crude extracts of wild type and PtoMYB240RNAi-pCXSN transgenic aspen leaf DNA;

8) About 1. Mu.l of RNase was added to the crude DNA extract, and RNA was digested at 37℃for 1 hour;

9) The DNA sample was stored in a-20deg.C refrigerator for further use.

(2) Positive plant PCR amplification

Because the wild plants do not contain exogenous transferred PCXSN carrier sequences, positive plant screening is performed by utilizing screening tag hygromycin specific amplification HYG-F and HYG-R primer amplification.

The specific primer sequences are as follows:

HYG-F：5’-ATCGGACGATTGCGTCGCATC-3’(SEQ ID NO.8)；

HYG-R：5’-GTGTCACGTTGCAAGACCTG-3′(SEQ ID NO.9)。

and taking a wild DNA template as a negative control, taking a vector plasmid which corresponds to the transgenic plant and has correct sequencing as a positive control, and carrying out PCR amplification and gel electrophoresis imaging on DNA of the transgenic plant to identify a transgenic positive strain. It was found that the target band of 0.5kb was amplified only in PtoMYB240RNAi-pCXSN plasmid DNA and L2, L3, L6, L7, L9 DNA. Successful transfer of PtoMYB240RNAi-pCXSN in PtoMYB240RNAi L2, L3, L6, L7, L9 plants was demonstrated (FIG. 2A).

And (5) screening by utilizing the amplification of the vectors HYG-F and HYG-R primers. The specific primer is designed and the specific primer is designed,

the PCR reaction system was as in Table 6, reaction procedure: pre-denaturation at 94℃for 3min,1 cycle; denaturation at 94℃for 30s, annealing for 30s, extension at 72℃for 1min for 31 cycles; extending at 72 ℃ for 10min; the amplified product was detected by 1% agarose gel electrophoresis.

TABLE 6 PCR reaction System

EXAMPLE 6 PtoMYB240RNAi-pCXSN transgenic plant expression level analysis

The experiment for identifying the gene expression level in RNAi interference plants requires the use of a fluorescent quantitative PCR method to quantitatively analyze the expression level of the corresponding genes. The fluorescent quantitative PCR adopts special DNA polymerase containing SYBR fluorescent dye, the dye can permeate into DNA double chains, so that the content of DNA is converted into a fluorescent value to be detected by an instrument, and the expression level of the corresponding gene in the template can be quantitatively calculated according to the fluorescent signal ratio after a certain cycle of PCR reaction.

The method comprises the following specific steps:

(1) Quantitative primer design

Specific primers are designed for genes to be detected, the amplification length is controlled to be 100-200bp, and the accuracy can be improved by crossing introns among the primers; the specific primers are as follows:

qPtoMYB240-F:5’-CAAGAACGTGGTGATCTAATCT-3’(SEQ ID NO.10)；

qPtoMYB240-R:5’-TTTCATCCCAGAGCCATT-3’(SEQ ID NO.11)；

(2) Quantitative PCR system and reaction conditions

The quantitative PCR system is shown in table 7:

TABLE 7 quantitative PCR System

Reaction conditions:

first, pre-denaturation at 95 ℃ for 30s;

secondly, denaturing for 5s at 95 ℃;

thirdly, annealing at 60 ℃ and extending for 1min;

fourth, the second to third steps are circulated for 40 times;

fifth, dissolution profile was 95℃for 15s,60℃for 30s, and 95℃for 15s.

The results are shown in FIG. 2B, where the expression levels of L2 and L3 were significantly reduced in PtoMYB240RNAi identified plants compared to WT.

EXAMPLE 7 PtoMYB240RNAi plant phenotyping of Populus tomentosa

Transplanting the tissue culture seedling of one month into a flowerpot, and growing for three months in a greenhouse under a long sunlight condition (16 hours of illumination/8 hours of darkness and 10000lux of light intensity) at 25 ℃. The plant height and stem thickness and dry weight of WT, ptoMYB240RNAi-pCXSN transgenic poplar were determined and counted.

The results are shown in FIG. 3A, where PtoMYB240RNAi L2 and L3 plants have significantly increased stem thickness (FIG. 3, B) and dry weight (FIG. 3, C) compared to WT plants.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. The application of interfering the expression of a transcription factor PtoMYB240 of populus tomentosa in improving the biomass of populus tomentosa is characterized in that: the nucleotide sequence of the transcription factor PtoMYB240 is shown in SEQ ID NO. 1.

2. Use of the interfering transcription factor PtoMYB240 of populus tomentosa according to claim 1 for increasing populus tomentosa biomass, characterized in that: the biomass is crude stem or dry weight.

3. Use of the interfering transcription factor PtoMYB240 of populus tomentosa according to claim 1 for increasing populus tomentosa biomass, characterized in that: one method of interfering with the expression of the populus tomentosa transcription factor PtoMYB240 is by RNAi technology.

4. Use of the interfering transcription factor PtoMYB240 of populus tomentosa according to claim 1 for increasing populus tomentosa biomass, characterized in that: the method for interfering the expression of the transcription factor PtoMYB240 of the populus tomentosa converts a recombinant vector containing RNAi fragments of the transcription factor PtoMYB240 into populus tomentosa, and screens transgenic plants.

5. Use of the interfering transcription factor PtoMYB240 of populus tomentosa according to claim 1 for increasing populus tomentosa biomass, characterized in that: the nucleotide sequence of the RNAi fragment is shown as SEQ ID NO. 2.

6. A method for improving the biomass of populus tomentosa, which is characterized by comprising the following steps: transforming the recombinant vector containing RNAi fragment of the transcription factor PtoMYB240 into populus tomentosa, and screening the transgenic plant to obtain the transgenic plant which is the populus tomentosa with improved biomass.

7. The method according to claim 6, wherein: the nucleotide sequence of the RNAi fragment of the transcription factor PtoMYB240 is shown as SEQ ID NO. 2.

8. The method according to claim 6, wherein: the transformation is agrobacterium-mediated.

9. The method according to claim 6, wherein: the agrobacterium is agrobacterium GV3101.